Poxviruses replicate in the cytoplasm of the cell and encode enzymes and factors needed for transcription of their genomes. Vaccinia virus, therefore, provides a unique system for combining biochemical and genetic approaches for investigating mechanisms of gene regulation and mRNA biosynthesis. Studies with vaccinia virus indicated that the genes are divided into three temporal classes - early, intermediate and late - that are regulated in a cascade fashion. The sites of interaction of the 82- and 70-kDa subunits of the early transcription factor (VETF) with the promoter were determined using DNA that was modified by the incorporation of aryl azide residues. We found that the 82-kDa subunit was cross-linked primarily with the core region of the promoter while the 70-kDa subunit interacted with the downstream region. Mutations in core region resulted in the loss of binding specificity. To investigate additional roles of VETF, we constructed a conditionally lethal recombinant vaccinia virus in which the D6R gene, encoding the 70-kDa subunit of VETF, is under stringent E. coli lac operator control. Electron microscopy revealed that immature virus particles and masses of electron dense material accumulated in the absence of inducer. We considered that VETF has a direct role in virion morphogenesis in addition to serving as an early transcription factor. The 26-kDa protein encoded by the vaccinia virus A2L open reading frame was shown to be required for in vitro transcription of a template with a late promoter and named late transcription factor 3 (VLTF-3). A fourth late transcription factor (VLTF-4) was purified to homogeneity from vaccinia virus infected HeLa cells and shown to be a product of the H5R open reading frame. Both VLTF-3 and VLTF-4 were expressed with a baculovirus vector and shown to be active in transcription assays.